Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.     

Characterization of β‐Cell Mass and Insulin Resistance in Diet‐induced Obese and Diet‐resistant Rats

The selectively bred diet‐induced obese (DIO) and diet‐resistant (DR) rats represent a polygenetic animal model mimicking most clinical variables characterizing the human metabolic syndrome. When fed a high‐energy (HE) diet DIO rats develop visceral obesity, dyslipidemia, hyperinsulinemia, and insulin resistance but never frank diabetes. To improve our understanding of the underlying cause for the deteriorating glucose and insulin parameters, we have investigated possible adaptive responses in DIO and DR rats at the level of the insulin‐producing β‐cells. At the time of weaning, DR rats were found to have a higher body weight and β‐cell mass compared to DIO rats, and elevated insulin and glucose responses to an oral glucose load. However, at 2.5 months of age, and for the remaining study period, the effect of genotype became evident: the chow‐fed DIO rats steadily increased their body weight and β‐cell mass, as well as insulin and glucose levels compared to the DR rats. HE feeding affected both DIO and DR rats leading to an increased body weight and an increased β‐cell mass. Interestingly, although the β‐cell mass in DR rats and chow‐fed DIO rats appeared to constantly increase with age, the β‐cell mass in the HE‐fed DIO rats did not continue to do so. This might constitute part of an explanation for their reduced glucose tolerance. Collectively, the data support the use of HE‐fed DIO rats as a model of human obesity and insulin resistance, and accentuate its relevance for studies examining the benefit of pharmaceutical compounds targeting this disease complex.     

我国8个典型水稻土中产甲烷古菌群落组成的空间分异特征

【目的】产甲烷古菌主导稻田甲烷生成,是稻田生态系统的模式微生物类群之一,具有重要的生态学意义。然而,水稻土产甲烷古菌群落组成的空间分异却鲜有报告。【方法】本研究沿20.55°N至47.43°N梯度,采集了我国不同纬度上8个典型水稻土,利用PCR-DGGE指纹图谱和系统发育树分析揭示不同地点水稻土中产甲烷古菌群落的组成;结合多个环境因子,利用生物信息学,典范对应分析(Canonical Correspondence Analysis,CCA)和维恩图(Venn diagram)明确产甲烷古菌的空间分异规律。【结果】研究发现p H值是驱动水稻土中产甲烷古菌群落组成分异的主要因子;此外,沿纬度梯度,8个地区的产甲烷古菌群落组成也呈现出规律性变化。【结论】本研究首次阐明了稻田中产甲烷古菌群落分布情况,并揭示其主要驱动因子。该认知不仅有助于我们更好地了解产甲烷古菌的生物地理学分布,还有助于从微生物学机制上阐明我国温度梯度带上有机质转化空间的差异。     

Shifts between Nitrospira‐ and Nitrobacter‐like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite‐oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter‐ and Nitrospira‐like NOB (by quantitative PCR) and the community structure of the Nitrobacter‐like NOB (by PCR‐DGGE and cloning‐sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no‐tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no‐tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization – i.e. ammonification – measured by the ¹⁵N dilution technique) were also surveyed. PNO was always higher for the no‐tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter‐like NOB and was also strongly related to gross mineralization, a proxy of N‐availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira‐like NOB. Selection of a dominant population was observed under no‐tillage, and PNO was loosely correlated to the community structure of Nitrobacter‐like NOB. Our results demonstrate that Nitrobacter‐like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira‐like and Nitrobacter‐like NOB and to a weaker extent by shifts of populations within Nitrobacter‐like NOB.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Diet‐induced Changes in Intra‐abdominal Adipose Tissue and CVD Risk in American Women

The aim of the study was to determine what effect weight loss had on intra‐abdominal adipose tissue (IAAT) and cardiovascular disease (CVD) risk in 135 premenopausal overweight African‐American (AA) and European‐American (EA) women matched for BMI. Blood lipids, systolic blood pressure (SBP), diastolic BP (DBP), and IAAT (computed tomography determined) were examined prior to and after an 800 kcal/day diet producing 12 kg‐weight loss. Significant decreases in IAAT (～38%), total cholesterol (TC; 3%), low‐density lipoproteins (LDLs: 6%), triglycerides (TGs: 27%), cholesterol/high‐density lipoprotein ratio (C/HDL ratio: 18%), SBP (3%), and DBP (3%) occurred while HDL increased (16%), following weight loss and 1 month energy balance. Significant interactions between time and race showed that AA women decreased TG and increased HDL proportionately less than EA women. After adjusting for ΔIAAT, none of the CVD variables significantly changed after weight loss with the exception of HDL and C/HDL ratio. After adjusting for ΔLF (leg fat), ΔTC, ΔTG, ΔLDL, and ΔC/HDL ratio were significantly different. Multiple regression showed that independent of each other, ΔIAAT was significantly and positively related to ΔTC (adjusted β = 0.24) and ΔTG (adjusted β = 0.47), and ΔLF was negatively related to ΔTC (adjusted β = ?0.19) and ΔTG (adjusted β = ?0.18). Overweight and premenopausal AA and EA women benefitted from weight loss by decreasing IAAT and improving CVD risk. The changes in IAAT were significantly related to blood lipids, but loss of LF seems to be related to reduced improvement in TC and TG. Based on these results, interventions should focus on changes on IAAT.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

Dynamics of the redox potential and rh of the rumen fluid of goats

The redox potential (Eh) of the rumen fluid of goats varied from -145 to -190 mV and the corresponding rH values from 6.3 to 8.6. The redox potential values of the rumen fluid were influenced by changes in pH. The most oxidizing Eh values --and at the same time the lowest rH and pH values--were observed after a feeding ration containing readily available carbohydrates. No relationship was found between the fermentation rate and the redox potential. The association between the oxidation reduction state and the metabolic activity is best expressed by the rH values. In in vitro experiments, a higher pH or the addition of cysteine or sodium sulphide moved the redox potential of the rumen fluid towards more reducing values. A shift towards more oxidizing values occurred after acidification of the medium, or after the action of heavy metal ions or atmospheric O2. Various other compounds, including bubbling of the rumen fluid with hydrogen, had little or no effect. SH-groups probably play an important role in the formation of the negative redox potential in rumen fluid.     

Disturbance‐induced changes in a high‐alpine cushionfield community,south‐central New Zealand

Long‐term effects of two nearby disturbances, mechanical blading 34 years ago and an approximately century‐old sheep graveyard, in high‐alpine cushionfield share certain vegetation features despite their contrasting soil nutrient status. Secondary succession following blading has continued beyond that recorded over the first 24 years with continued trends towards the adjacent undisturbed cushionfield: increased cover of several cushion species and the lichen Cetraria islandica ssp. antarctica. Several mid‐seral species remain prominent while bare soil and stone pavement continue to decrease. Additional shelter, facilitated by tall snow tussocks planted locally at near‐natural density on part of the bladed site, allowed establishment of some larger, thin‐leaved species. Soil factors remain little affected by the mechanical disturbance compared with the general enrichment persisting at the graveyard. Here three small indigenous grasses, which dominated 50 years earlier (Trisetum spicatum, Agrostis muelleriana, Poa colensoi), remain prominent among the relatively diverse community (50 species, including several calciphilic lichens on the degrading bones). By contrast, frequency of the cushionfield dominant, Dracophyllum muscoides and the lichen Alectoria nigricans, are negatively correlated with the presence of bones. The post‐disturbance temporal community changes are assessed in the context of the initial productivity of the habitat, competitive exclusion and the intermediate disturbance hypothesis.     

Diet‐induced obesity impairs AKT signalling in the retina and causes retinal degeneration

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high‐fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT₁, eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4‐hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin‐resistance‐induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes. Copyright © 2012 John Wiley & Sons, Ltd.     

Diet‐induced Obesity Delays Cardiovascular Recovery from Stress in Spontaneously Hypertensive Rats

Objective: Blood pressure (BP) and heart rate (HR) responses to stress are significant predictors of cardiovascular morbidity and mortality. Because obesity is a major risk factor for cardiovascular disease, we examined whether diet‐induced obesity alters the BP and HR responses to stress and whether these alterations are associated with augmented cardiovascular morbidity in the rat. Research Methods and Procedures: Adult male spontaneously hypertensive rats were fed either a normal diet or high‐fat diet (HFD) for 12 weeks. At weeks 0 and 12, body weight was measured, and BP and HR were recorded by radiotelemetry throughout three consecutive day and night periods and in response to 30‐minute immobilization stress. At the end of the 12‐week intervention, the rats were sacrificed, and their organs and sera were collected. Results: With the intervention, HFD rats showed a significantly greater increase in body weight (as expected) and circulating leptin and free fatty acid levels compared with normal diet rats. In addition, they showed similar increases in BP and HR elevations during stress but significantly slower BP and HR decreases after stress. These HFD‐induced delays in stress recovery were associated with BP and HR elevations during the night (behaviorally active) period and with augmentations in cardiac mass. Discussion: The results of this study indicate that, in spontaneously hypertensive rats, dietary obesity delays cardiovascular recovery from stress, and, in parallel, it promotes the development of nocturnal hypertension as well as cardiac hypertrophy. This suggests that dietary obesity may significantly potentiate the impact of daily stressful experiences on the cardiovascular system.     

Interaction of Leptin and Amylin in the Long‐term Maintenance of Weight Loss in Diet‐induced Obese Rats

We have previously shown that combined amylin + leptin agonism elicits synergistic weight loss in diet‐induced obese (DIO) rats. Here, we assessed the comparative efficacy of amylin, leptin, or amylin + leptin in the maintenance of amylin + leptin–mediated weight loss. DIO rats pretreated with the combination of rat amylin (50 µg/kg/day) and murine leptin (125 µg/kg/day) for 4 weeks were subsequently infused with either vehicle, amylin, leptin, or amylin + leptin for an additional 4 weeks. Food intake, body weight, body composition, plasma parameters, and the expression of key metabolic genes in liver and white adipose tissue (WAT) were assessed. Amylin + leptin treatment (weeks 0–4) reduced body weight to 87.5% of baseline. Rats subsequently maintained on vehicle or leptin regained all weight (to 104.2 and 101.2% of baseline, respectively), those maintained on amylin had partial weight regain (97.0%). By contrast, weight loss was largely maintained with continued amylin + leptin treatment (91.4%), associated with a 10% decrease in adiposity. Cumulative food intake (weeks 5–8) was reduced by amylin and amylin + leptin, but not by leptin alone. Amylin + leptin, but not amylin or leptin alone, reduced plasma triglycerides (by 55%), total cholesterol (by 19%), and insulin (by 57%) compared to vehicle. Amylin + leptin also reduced hepatic stearoyl‐CoA desaturase‐1 (Scd1) mRNA, and increased WAT mRNA levels of adiponectin, fatty acid synthase (Fasn), and lipoprotein lipase (Lpl). We conclude that, in DIO rats, maintenance of amylin + leptin–mediated weight loss requires continued treatment with both agonists, and is accompanied by sustained improvements in body composition, and indices of lipid metabolism and insulin sensitivity.     

Characterization of start-up performance and archaeal community shifts during anaerobic self-degradation of waste-activated sludge

Successful start-up strategy for anaerobic digestion of waste-activated sludge using internal inoculum and relationship between the shift of methanogenic community and the digester performance during start-up was investigated. Combination of TS control of inoculum and batch operation during early days enabled the successful start-up operation without serious volatile fatty acid accumulation, followed by the stable continuous operation. However, the propionate degradation was rate-limiting step during the batch operation. The results of real-time quantitative polymerase chain reaction analysis suggested that there was a correlation between the population of the genus Methanosarcina and the methane production rate coupled with acetate consumption during batch operation, and the results of terminal-restriction fragment length polymorphism (T-RFLP) revealed that the increasing intensity of T-RF peaks of hydrogenotrophic methanogens was associated with a decrease in the level of C3-acids.     

High‐fat Diet‐induced Ultradian Leptin and Insulin Hypersecretion are Absent in Obesity‐resistant Rats*

Objective: Sprague‐Dawley rats fed a high‐fat diet (HFD) are either obesity prone (OP) or obesity resistant (OR). We tested the hypothesis that differences in the ultradian rhythmic patterns of insulin and ghrelin in OP vs. OR rats promote obesity in OP rats. Research Methods and Procedures: Rats were fed regular chow or an HFD, and ultradian fluctuations in leptin, insulin, and ghrelin were analyzed in blood samples collected at 5‐minute intervals from intrajugular cannulae of freely moving rats. Results: Regular chow feeding resulted in a slow weight gain accompanied by small increases in insulin and leptin and a decrease in ghrelin discharge, with only the pulse amplitude significantly altered. Similar changes were observed in OR rats, despite HFD consumption. In contrast, OP rats exhibited a high rate of weight gain and marked hyperinsulinemia, hyperleptinemia, and hypoghrelinemia; amplitude was altered, but frequency was stable. In a short‐term experiment, HFD elicited similar secretory patterns of smaller magnitude even in the absence of weight gain. Discussion: We showed that three hormonal signals of disparate origin involved in energy homeostasis were secreted in discrete episodes, and only the pulse amplitude component was vulnerable to age and HFD consumption. Increases in insulin and leptin and decreases in ghrelin pulse amplitude caused by HFD were exaggerated in OP rats relative to OR rats and preceded the weight increase. These findings show that a distinct genetic predisposition in the endocrine organs of OR rats confers protection against high‐fat intake‐induced ultradian hypersecretion of obesity‐promoting hormonal signals.     

Diet‐induced early‐stage atherosclerosis in baboons: Lipoproteins,atherogenesis, and arterial compliance

Background

The purpose of this study was to determine whether dietary manipulation can reliably induce early‐stage atherosclerosis and clinically relevant changes in vascular function in an established, well‐characterized non‐human primate model.

Methods

We fed 112 baboons a high‐cholesterol, high‐fat challenge diet for two years. We assayed circulating biomarkers of cardiovascular disease (CVD) risk, at 0, 7, and 104 weeks into the challenge; assessed arterial compliance noninvasively at 104 weeks; and measured atherosclerotic lesions in three major arteries at necropsy.

Results

We observed evidence of atherosclerosis in all but one baboon fed the two‐year challenge diet. CVD risk biomarkers, the prevalence, size, and complexity of arterial lesions, plus consequent arterial stiffness, were increased in comparison with dietary control animals.

Conclusions

Feeding baboons a high‐cholesterol, high‐fat diet for two years reliably induces atherosclerosis, with risk factor profiles, arterial lesions, and changes in vascular function also seen in humans.     

Cadmium‐induced and trans‐generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana

Trans‐generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well‐known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd‐exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd‐exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.     

Roux‐en‐Y Gastric Bypass Enhances Energy Expenditure and Extends Lifespan in Diet‐induced Obese Rats

Gastrointestinal weight‐loss surgery (GIWLS) is currently the most effective treatment for severe obesity, with Roux en‐Y gastric bypass (RYGB) among the best of the available surgical options. Despite its widespread clinical use, the mechanisms by which RYGB induces its profound weight loss remain largely unknown. This procedure effects weight loss by altering the physiology of weight regulation and eating behavior rather than by simple mechanical restriction and/or malabsorption as previously thought. To study how RYGB affects the physiology of energy balance, we developed a rat model of this procedure. In this report, we demonstrate that RYGB in diet‐induced obese (DIO) rats induces a 25% weight loss, prolongs mean survival by 45%, and normalizes glucose homeostasis and lipid metabolism. RYGB induced a 19% increase in total and a 31% increase in resting energy expenditure (REE). These effects, along with a 17% decrease in food intake and a 4% decrease in nutrient absorption account for the normalization of body weight after this procedure. These effects indicate that surgery acts by altering the physiology of weight regulation and help to explain the effectiveness of RYGB in comparison to restrictive dieting and other forms of dietary and pharmacological therapies for obesity. The clinical effectiveness of RYGB and its physiological effects on body weight regulation and energy expenditure (EE) suggest that this operation provides a unique opportunity to explore the mechanisms of energy homeostasis and to identify novel therapies for obesity and related metabolic diseases.